The long-term focus of this grant continues to be the structure and function of the visual pigment rhodopsin and its interactions with other members of the vertebrate phototransduction cascade. The studies are geared toward an understanding of the protein in terms of its mechanism of activation, its interaction with downstream proteins of the phototransduction cascade, and its function and dysfunction in health and disease. The new studies focus on two areas: the development and exploitation of a single-molecule approach to the study of interactions among signaling partners in the visual phototransduction cascade, and the characterization of novel mutants of rhodopsin in which the active site of the protein has been significantly altered from the wild-type. There are two Specific Aims: 1. to establish, characterize, and expand Total Internal Reflection Fluorescence Microscopy (TIRFM) as a tool in the single-molecule regime to study the dynamics of assembly and disassembly of key signaling complexes in the rod cell phototransduction cascade. One of the major goals of this aim is to better understand the role of constitutively active mutants in the diseases retinitis pigmentosa and congenital stationary night blindness. 2. To characterize novel rhodopsin mutants in which the highly conserved active-site Lys296 has been moved to different locations in the protein. This Aim expands upon surprising preliminary results showing that the Lys can be moved while maintaining near wild-type spectral properties and ability to activate transducin in a light-dependent manner. The focus is to better understand the evolutionary relatedness of retinylidene proteins and to probe our understanding of the molecular mechanism of photoactivation of rhodopsin.